Osteo-lineage cells are of particular importance in the life-long regenerative processes of bone. They are also critical in the bone marrow microenvironment where they control hematopoiesis and likely participate in leukemia and cancer metastasis. However, despite the pivotal roles of osteogenic cells in the maintenance of bones and hematopoiesis, the in vivo origin, lifespan, and dynamics of these cells remain fundamentally unanswered questions. Therefore, we aimed to define the lifespan of bone-forming osteoblasts and their source of replacement under homeostatic and injury conditions. Our recent studies using genetic pulse-chase techniques in combination with time-lapse live-animal fluorescent imaging have revealed that functional osteoblasts and osteoblast precursors have a short lifespan and are durably maintained by their progenitor cells in vivo. Additionally, we found that the 'mesenchymal stem cells' (as defined by in vitro multilineage capacity) have no such function in vivo. Rather, they serve as osteolineage-restricted progenitors in normal homeostasis. These osteogenic progenitors can be genetically marked by transient activation of the myxovirus resistance-1 (Mx1) promoter. We demonstrated that Mx1-labeled osteogenic progenitor cells have a perivascular origin, migrate efficiently to sites of injury, and supply the majority of new osteoblasts in fracture healing. Further, they overlap with a subset of perivascular nestin MSCs. We now have distinct stage-specific inducible models that can label osteogenic stem/progenitors, pre-osteoblasts and mature osteoblasts. The goal of this study is to understand the role of mature osteoblasts and their stem/progenitor cells in the context of bone regeneration and repair in vivo. We will also explore the clinical relevance of osteogenic stem cells with the goal of reversing conditions of degenerative bone disease. We thus propose to: 1. Test if Mx1 and nestin double labeling specify osteogenic stem cells in vivo. 2. Test if te transplantation of osteogenic stem cells improves the restoration of bone fracture. 3. Test the role of osteogenic stem cells in bone loss diseases. By studying three specific, clinically relevan questions, we will further define new biological and clinical insights of osteogenic stem cells in physiological conditions. PUBLIC HEALTH RELEVANCE: Mesenchymal cell kinetics are relevant for most tissues and of particular importance in bone and bone marrow where skeletal mesenchymal cells play critical roles in regulating regenerative processes and disease progression. Bone matrix is known to turnover rapidly, but the dynamics of bone producing and remodeling cells are less well defined. We recently examined the lifespan of bone-forming osteoblasts and their source of replacement under homeostatic and injury conditions. Using newly developed genetic pulse-chase models in combination with sequential intravital microscopy, we discovered a short lifespan and unexpected dynamics of osteoblasts in vivo. Additionally, we found a population of progenitors that have the multilineage potential of MSC in vitro but have no such function in vivo. Rather, this population serves as osteolineage-restricted stem/progenitors in normal homeostasis and after bone injury. Therefore, we seek to examine the role of mature osteoblasts and their stem cells in controlling tissue regeneration and repair in physiologic circumstances. We also propose to examine the clinical benefit of osteogenic stem cells upon transplantation in bone disease conditions. The proposed experiments will elucidate: (1) the in vivo characterization of osteogenic stem cells, (2) the recovery of bone fracture by transplantation of osteogenic stem cells, and (3) the role of osteogenic stem cells in bone loss diseases. Overall, these three clinically relevant aims will further define new biological and clinical insights of osteogenic stem cells in physiological conditions.